Antioxidizing composition for scavenging free radicals, pharmaceutical composition comprising the same, and process for preparing the same

ABSTRACT

An anti-oxidizing composition for scavenging free radicals, comprising at least one essential oil component containing a number of fat-soluble, low-molecular-weight compounds, a pharmaceutical composition comprising the above composition, and a process for preparing the same are described. Percutaneous absorption of the pharmaceutical composition is effective in treating immune diseases, particularly AIDS.

This application is a 35 U.S.C. §371 of PCT/JP97/03446, filed Sep. 26,1997.

FIELD OF THE INVENTION

The present invention relates to an antioxidizing composition forscavenging free radicals generated in a living body, a pharmaceuticalcomposition comprising thereof, and a process for preparing thereof.

BACKGROUND OF THE INVENTION

Active oxygen or free radical causes various troubles in a living body.Active oxygen or free radical is generated by bacteria invasion into theliving body, in chitin-chitosan oxidase system, arachidonic cascadesystem, or a process of saccharification, and so forth.

Active oxygen is a generic name of super hydroxy radical (.O₂ ⁻),singlet oxygen (¹O₂) and hydroxy radical (.OH). Hydroxy radical isformed by taking an electron into the orbital of triplet oxygen inground states. The singlet oxygen is defined as the oxygen wherein twounpaired electrons of an oxygen atom make a pair to enter orbital of theother oxygen atom, resulting the electron orbitals are empty.

Furthermore, perhydoxyradical (.OOH), which is a typical free radical,is generated when peroxide is decomposed by autoxidation via thefollowing route.

RH+O₂→ROOH→R.+.OOH

Perhydoxyradical looses a proton in alkaline solution to be converted tosuperoxide (.OO⁻). In the living body, free dihydroflavinnon-enzymatically reacts with oxygen very quickly to generate perhydroxyradical.

Perhydroxyradical is converted to the above-mentioned superoxide, andthen superoxide is catalyzed by superoxide dismutase (SOD) to generatean oxygen molecule and hydrogen peroxide.

2.O₂ ⁻+2H⁺⇄O₂+H₂O₂

In the bacteria invasion, free radicals including active oxygen aregenerated to exclude extraneous material for the living body; afterbacteria invade to the living body, neutrophils phagocytose suchmaterials and release activated oxygen from their membrane. Inchitin-chitosan oxidase system, serious tissue lesion occurs; inischemia, active oxygen is generated by increasing hypoxanthine andxanthine oxidase level in cells, and after ischemia a larger amount ofactive oxygen is generated by introducing a lot of oxygen into thecells. Such tissue lesion is referred to as ischemia-perfusion disorder.In an arachidonic acid cascade system, unsaturated fatty acid isperoxidized through autoxidation mechanism to generate peroxides andradicals.

Since thus generated free radicals cause various diseases such asinflammation, allergy, cerebral nerve disease and tissue lesions, it isrequired that the excessive generation of free radicals should beprevented or the generated free radicals should be scavenged. Targetmolecules and lesions caused by damaging them are shown in Table 1.

TABLE 1 In vivo target molecule for active oxygen and free radicalsTarget molecules Lesions Lipids Peroxidation and lesion of biomembraneNucleic Acids Cell cycle change, mutation, DNA chain cleavage, basedamage, carcinogenesis, and AIDS Amino Acids Denaturation of protein,polymerization, enzyme repression, and cross-linking denaturationCarbohydrate Change of cell surface receptor Hyaluronic acidDepolymerization Biological Factors Inactivation (α-antitrypsin,chemotactic factor, chemical mediator, neurotransmitter)

Among the substances, which are biosynthesized in vivo, any substancecapable of scavenging .OH is not known. In vivo, .OH is generated byreduction of H₂O₂ by using transition metal, decomposition of H₂O uponirradiation and reaction of NO with superoxide. This .OH is highlyreactive and reacts with almost all the biosubstance in arate-determining manner by nucleic acids. Therefore, the .OH often givesa living body a mortal wound. Accordingly, in order to prevent orterminate such wounds, it is required to provide a substance havingantioxidation activity for .OH from outside of the living body.

As active oxygen inhibitors, which are presently known, there can bementioned ceruloplasmin, metallothionein and vitamin C. Ceruloplasmin isa plasma protein of which molecular weight is about 16,000, including 6to 7 copper ions per molecule, and it is known that it shows SODactivity against .O₂ ⁻. This protein is rich in blood, and it seems tobe a scavenger of active oxygen (.O₂ ⁻) in blood vessel. Metallothioneinis a metalloprotein of which molecular weight is about 60,000, andcysteine occupies 30% of its structure protein. It is known that thisprotein scavenges hydroxy radicals (.OH) and suppresses peroxidation oflipids. Vitamin C is known as a scavenger for .O₂ ⁻, .OH, singlet oxygen(¹O₂), hydrogen peroxide (H₂O₂), and so forth. These substances are usedas pharmaceuticals, but all of them are water-soluble and have arelatively high molecular weight. Furthermore, they are mainlyadministered per os or via injection. They act as the scavengers inextracellular fluid, but are not incorporated into the cells.

In general, drugs are transferred into blood and then delivered totarget sites by blood flow. Since fat-soluble drugs pass throughblood-brain barrier (BBB) to be transferred into brain, such drugs havea trouble from the viewpoint of side effects. Therefore, water-solubledrugs are selected, and such administration routes are used. However, itis difficult to deliver the drug to the target sites with effective doseby transferring the drug in blood. Particularly, it is almost impossibleto deliver the drug at such dose when the target site is lymph tissue.

Such drugs are desired to have a low molecular weight rather than a highmolecular weight from the viewpoint of formulation or administration,and to be compounds or compositions having antioxidizing activity. Asmentioned above, free radicals cause various diseases or tissue lesions,particularly peroxidation of lipid, to generate peroxides and radicalsto influence badly for living body. Considering these matters,lymptropic drugs or compositions are needed.

However, lymptropic drug or compositions having antioxidizationactivities are not known. Particularly, those capable of preventinglipid peroxidization have not found.

On the other hand, it is known that enzymes such as SOD, catalase andglutathione peroxidase scavenge free radicals. Furthermore, as examplesof antioxidizing substances, which are defined as the substances havingantioxidization activities, a singlet oxygen scavenger, a superoxidescavenger, a hydroxy radical scavenger, and an antioxidizing of whichmechanism is not clarified are known. Specifically, as examples of thesinglet oxygen scavenger, there can be mentioned β-carotene, amines,tocopherols and histidines; as superoxide scavengers, tocopherols,phenols, thiols, ascorbic acid, and copper(II)-histidine complexes; ashydroxy radical scavengers, mannitol; and as the antioxidizing of whichmechanism is not clarified, nucleic acid bases, fulavonoids, sterol,terpene, and polycarboxilic acid.

Accordingly, there are not effective means to scavenge free radicals atpresent, and only preventive means for free radical generation is used.In such preventive means, enzymes such as SOD, catalase and glutathioneperoxidase, fat-soluble or water-soluble vitamins included in foods suchas vitamin E, which is called tocopherol, vitamin A, vitamin D, vitaminC and vitamin Bs, and various coloring matters such as carotenoid whichis fat-soluble coloring matters, and fulavonoid which is water-solublecoloring matters are taken parenterally or per os as antioxidizingsubstances.

Such compounds may be taken alone, or in combination; for example,several antioxidizing compounds such as tannin, flavonoid, carotenoid,vitamin C, caffeic acid derivatives, lignans and saponin which areincluded in Chinese orthodox crude drug used in Oriental medicine fromancient age.

In Chinese orthodox medicine, it is known that those antioxidizingcompounds play great roles in action mechanism. However, since thesecompounds are water-soluble, there is a problem from the viewpoint oflymptropic activity.

When the above-mentioned antioxidizing enzymes or substances are takenper os, those enzymes are digested digestive tract or liver inabsorption step to be metabolites, their bioavailability as drugs arenot enhanced. Furthermore, if they are administrated parenterally, theyare transferred into blood but not into lymph. Such tendency is alsoobserved in the antioxidizing substance, vitamins described above.

SUMMARY OF THE INVENTION

Inventors of the present invention have studied eagerly to solve theproblems mentioned above. They found that essential oil componentincluding fat-soluble compounds with a low molecular weight haveantioxidizing activity and at least one component in such essential oilsare transferred into lymph at a high ratio when they are adsorbedpercutaneously, and completed the present invention.

The first embodiment of the present invention is a free radicalscavenging composition comprising at least one essential oil including aplurality of fat-soluble low-molecular-weight compounds, which ischaracterized as having antioxidization activity. The fat-solublecompounds are a straight or branched chain compound with 4 to 14 carbonatoms. The fat-soluble low-molecular-weight compound is selected fromthe group consisting of phenolic compounds from naturally occurringsubstance, sulfur-containing compounds, and terpenoid. Furthermore, thephenolic compounds are selected from the group consisting of thyme oil,an essential oil from Labiatae plants and that from Eucalyptus plants.Sulfur-containing compound is selected from the group consisting ofgarlic oil, welsh onion oil and scallion oil. The terpenoid is selectedfrom the group consisting of lemon oil, an essential oil from Rutaceaeplants and lavender oil.

The second embodiment of the present invention is a pharmaceuticalcomposition containing the antioxidizing compositions described above.The pharmaceutical composition comprises the antioxidizing compound, apolymer resin, finely divided activated carbon particles, a poroussubstance, and a thermoplastic resin. The composition of the presentinvention may further comprise a coloring agent, a polysaccharide, awater-soluble resin, and so forth. The polymer resin is preferably anacrylic polymer having water absorption properties. The porous substanceis preferably zeolite, and its pore size is preferably in the range ofabout 3 to about 10 Å. The coloring matter is preferably that whichabsorbs light with a wavelength in the range of 400 to 450 nm. Thepolysaccharide is preferably chitosan or cellulose. The thermoplasticresin is preferably polyethylene, and the water-soluble resin ispolyvinyl alcohol (PVA).

In the second embodiment of the present invention, the pharmaceuticalcomposition is inserted between two pieces of sheet materials. Then thewhole edges of the pieces are bonded by heat pressing each other to forma percutaneous absorption agent. The sheet material is preferably madeof paper or non-woven fabric. The pharmaceutical composition ispreferably used for immune diseases, and especially for AIDS orautoimmune disease. Furthermore, the composition of the presentinvention is a composition applying for oxidation disorder.

The third embodiment of the present invention is a process for producinga pharmaceutical composition comprising the steps of adsorbing theantioxidizing composition onto the surface of each thermoplastic resinbead, forming an oil film on the antioxidizing composition-adsorbedresin surface, adsorbing finely divided carbon powder particles on thefilm-formed to form a capsule, preparing a base material by mixingtogether a polymer resin, a porous substance and a poly saccharide, andthen mixing together the capsule, the base, and a coloring agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the change of CD4⁺ and CD8⁺ T cell number whenthe pharmaceutical composition of the present invention or placebo isadministrated to cats appearing AIDS symptoms.

As shown in FIG. 1, the CD4⁺ and CD8⁺ T cell numbers are increasedquickly after administrating the present composition. When theadministration of the present composition is stopped, both the cellnumbers are decreased. However, the cell numbers are again greatlyincreased when the composition is administrated again.

FIG. 2 is a graph showing the suppression effect for oxidation disordercaused by methylene blue administration. The composition of theinvention and placebo are administrated to SPF cats by using gasformulation through the skin of back of cervix. Then methylene blue isadministrated at a dose of 1 mg/kg by every 8 hours, and Heinz bodyformation ratios in erythrocytes are compared after 40 hours from thebeginning.

The formation ratio of Heinz body is suppressed by administrating thepresent composition and there is a significant difference in the Heinzbody formation ratios as observed 40 hours after.

MODE FOR CARRYING OUT THE INVENTION

The present invention will now be explained in detail hereinbelow.

The anti-oxidizing composition of the present invention comprises atleast one essential oil component, which includes a plurality offat-soluble compounds having a low molecular weight. The fat-solublecompounds contain 4 to 14 carbon atoms, and it may contain hetero atoms.These fat-soluble compounds may have a straight chain, branched chain,or ring structure, and may have a heterocycle or fused ring when thefat-soluble compounds has a ring structure. The compound may besaturated or unsaturated, and has preferably a terpene structure when ithas a ring structure. The compound contains preferably a sulfur atomwhen it has a straight or branched chain structure. Such compoundshaving a low molecular weight is preferably on all of those contained inthe essential oil.

The essential oil is generic name of volatile oily compounds havingaroma and the essential oil is obtained by steam distillation fromvarious plants. In general, the essential oil is composed of manycomponents and it is neither fats nor fatty oil. The main components inthe essential oil are monoterpene, sesquiterpene, diterpene, and theiroxidized or reduced derivatives, but small amounts of higher terpenesand non-terpenes are contained in the essential oil. In general, theessential oil is insoluble in water but soluble in alcohol, etc. Aboutone thousand species of higher plants irregularly distributed in about60 families can produce them, but they are distributed concentratedly inPinales family, Umbelliferae family, Rhodomyrtus family and other coupleof families.

Extracts obtained by steam distillation and expression are referred toas the essential oil, and those obtained by extraction are given severalnames such as pomade, concrete, absolute, resinoid, oleoresin, tinctureand so forth depending on the particular extraction method.

As the essential oil employed in the present invention, there can bementioned Eucalyptus oil, thyme oil, Labiatae oil other than thyme oil,garlic oil, welsh onion oil, scallion oil and Rutaceae oil such as lemonoil.

Eucalyptus oil is obtained by steam distilling leaves of plantsbelonging to Eucalyptus, and is classified into four types. One iscinede type Eucalyptus oil obtained from Eucalyptus globulus Labill., ofwhich origin is Tasmania, and is mainly produced in north America,Mexico, Africa and south Spain. One of the others is piperitonephellandrene type Eucalyptus oil obtained from Eucalyptus dives Schauer,which is mainly produced in New South Wales and Victoria at Australia.The other one is geranyl acetate type Eucalyptus oil obtained fromEucalyptus macarthuri H. Deane et J. H. Maiden, which is mainly producedin southern area of New South Wales at Australia. The last one iscitronellal type Eucalyptus oil obtained from Eucalyptus citoriodoraHook, which is mainly produced in Queensland at Australia, South Africa,Brazil, Java, and India.

Cineole type Eucalyptus oil may be obtained from leaves of Eucalyptusglobulus Labill. by steam distillation, and contains the followingcomponents: cineole (about 70 to 80%), α-pinene, camphene, pinocarveol,pinocarvone, myatenol, berebenone, carvone, eudesmol, and C₄ to C₆aliphatic aldehyde, and the like.

Piperitone phellandrene type Eucalyptus oil may be obtained from leavesand branches of Eucalyptus dives Schauer by steam distillation, andcontains the following components: piperitone (about 40 to 50%),α-phellandrene (about 20 to 30%), p-cymene, camphene, dipentene,α-thujene, and the like.

Geranyl acetate type Eucalyptus oil may be obtained from leaves andbranches of Eucalyptus macarthuri H. Deane et J. H. Maiden by steamdistillation, and contains the following components: geranyl acetate(about 70%), geraniol (about 3%), eudesmol (about 16%), and otheraliphatic aldehydes.

Citronellal type Eucalyptus oil may be obtained from Eucalyptuscitoriodora Hook by steam distillation, and contains, for example,d-citronellal or l-citronellal (65 to 80%), d- or l-citronellal (15 to20%), phellandrene, cineole, citronellye acetate, geranyl acetate, andisopulegol.

Essential oil from Labiatae is generic name of perilla oil, Agastacherugosa O. Kuntze oil, clary sage oil, sage oil, Nepeta oil, Catnip oil,spearmint oil, pennyroyal oil, Rosemary oil, Basil oil, Lavandin oil,Lavender oil, and other oil. Perilla oil is obtained by steamdistillation from whole plant body from Perilla frutuescenes var. cripsaDecne. Froma viridis Makino, which is widely cultivated in Japan. Theoil contains, for example, perillaldehyde (40 to 55%) of which isinherent components, α-pinene, and limonene.

Agastache rugosa O. Kuntze oil is obtained by steam distillation fromwhole plants or flowers Agastache rugosa O. Kuntze cultivated intemperate zone of Asia, for example, China, Japan, and Taiwan. There isa slight difference among chemical components in the composition of oildepending on the producer, and the oil produced in Taiwan comprisesd-limonene, l-pulegone, l-isopulegone, other terpenoid, alcohol andester. The oil produced in Japan comprises, for example, methylchavicol, anisaldehyde, p-methoxy-cinnnamic aldehyde, 3-octanol,1-octene-3-ol, and other terpenoid.

Clary sage oil is obtained by steam distillation from whole plants orflowers of Salvia sclarea L. The oil comprises, for example, l-linalol,linalyl acetate, scrareol, and neroridol.

Sage oil is obtained by steam distillation of dried leaves of Salviaoffensinalis L., which are produced in Yugoslavia, Bulgaria, Turkey,France, Germany and North America. The oil comprises, for example,cineole, thujone, borneol, and camphor.

Nepeta oil and Catnip oil are obtained from the whole plants of Nepetacataria L. by steam distillation, and contains nepetaric acid,nepetalactone, nepetaric anhydride, β-caryophyllene, and the like.

Mint oil is classified into Japanese mint oil, peppermint oil, andspearmint oil and pennyroyal oil. Japanese mint oil is obtained by steamdistillation from the whole plants of Menta arvensis produced inHokkaido, Okayama, Brazil, Paraguay and China, and comprises l-menthol,menthone, menthyl acetate, thymol, sesquiterpenoid, lower fatty acidsand esters thereof.

Peppermint oil is obtained from the whole plants of Menta piperita var.vulgaris L. produced in north America, Italy, France, Bulgaria, Moroccoand others by steam distillation, and comprises l-menthol, menthylester, menthone, menthofuran, and other components which are almost thesame components as contained in Japanese mint oil.

Spearmint oil is obtained from the whole plants of Menta spicata Huds,var. tenuis (Michx) which is produced in north America, many areas ofEurope by steam distillation, and comprises, for example, 1-carvone,menthone, and pulgone.

Pennyroyal oil is obtained from the whole plants of Menta pulegium L.var. eriantha by steam distillation, and comprises, for example,d-pulegone, l-menthone, d-isomenthone, piperitone, piperitone, andisopiperitenone.

Patchouli oil is obtained from leaves of Pogostemon cablin Benth. bysteam distillation which is produced in for example, Philippines,Sumatra, and Borneo, and it comprises Patchouli alcohol, Patchoulion,Patchoulenone, cuminaldehyde and other terpenoid.

Rosemary oil is obtained from flowers, leaves or whole plants ofRosemarinus officinalis L. by steam distillation, and it comprises forexample, borneol, bornyl acetate, camphor, and cinede.

Basil oil is obtained from flowers or whole plants of Ocimum basilicumL. by steam distillation, and, depending on the inherent componentscontained in the oil, it is classified into: a methylchavicol typecontaining for example, methyl-chavicol as an inherent component,linalol, and an eugenol; methyl-cinnamate type containingmethylcinnamate as the inherent component, for example, methylchavicol,and linalol; an eugenol type containing eugenol as the inherentcomponents; a camphor type containing d-camphor as the inherentcomponent, a citroneral type containing citroneral as the inherentcomponent; and a thymol type containing thymol as an inherent component.

Lavandin oil is obtained by steam distillation from flowers of Lavendulahybrida Reverch which is produced in south France, and it comprises, forexample, linalol, linalyl acetate, linalol oxide, cinede, d-camphor, anddl-lavandulol.

Lavender oil is obtained by steam distillation from flowers of Lavendulaofficinalis Chaix. which is mainly produced in France, Italy, Hungary,southern part of ex USSR, England, north America and Australia, andcomprises, for example, linalol (10 to 20%), linalyl acetate (30 to60%), lavandulol, lavandulyl acetate, 3-octanol, α-pinene, β-pinene,limonene, cineole, and citronellal.

Several kinds of oil derived from Labiatae plants described above arepreferably used in combination, and more preferably two kinds of the oilis used in combination. Particularly, lavender oil is preferablyemployed.

Thyme oil is obtained by steam distillation from whole plant inflowering Thymus vulgaris L. or dried one which is produced in, forexample, south France, Italy, Turkey, Spain, Morocco and Israel, and itcomprises, for example, thymol (40 to 60%), p-cymene, carvacrol,linalol, geraniol, borneol, camphene, pinene, and caryophyllene.

Garlic oil is obtained by steam distillation from bulbs of Alium sativumL. which is produced in Europe, northern part of China and Japan, and itcomprises, for example, sulfur-containing compounds such asdiallylsulfide, diallyldisulfide, allylpropyldisulfide,diallyltrisulfide, and allicin.

Welsh onion oil is obtained by stem distillation from whole plants ofAllium fistulsum L. that is a kind of leek belonging to Liliaceaefamily. Scallion oil is obtained by steam distillation from whole plantsof Allium bakeri Regel. belonging to Liliaceae family and of whichorigin is China.

Rutaceae oil is generic name of lime oil, sweet orange oil, grapefruitoil, neroli oil, bergamot oil, mandarin orange oil, lemon oil, Japanesepepper oil and others.

Lime oil is obtained by expression as expressed lime oil from immaturefruits or mature fruits of Citrus aurantifolia Swing (acid lime). Whenpeel of acid lime is subjected to steam distillation, distilled lime oilis obtained. As the components contained therein, for example, citral,methylanthranilate, bisabolene, bergaptol, limonene, and aliphaticaldehyde such as n-nonylaldehyde are mentioned.

Sweet orange oil is obtained by expression from fruits of Cirus sinensisOsbeck var. brasiliensis Tanaka. The sweet orange oil and fruit juicecan be obtained separately. As the components contained therein, forexample, d-limonene, citral, n-decylaldehyde, d-linalol, d-terpineol,and n-nonylalcohol are mentioned, and the content of d-limonene is morethan 90%.

Grapefruit oil is obtained by expression from pericarp of Ctrus paradisiMacfayden (grapefruits) which is mainly produced in California, Florida,Texas, Israel and Brazil. Alternatively, when leaves or branches aresubjected to steam distillation, grapefruit petitgrain oil is obtained.As the components contained therein, d-limonene (more than 90%),nootkatone as the inherent component, octylaldehyde, citral, geranioland acetate ester thereof are mentioned.

Neroli oil is obtained by steam distillation from flowers of Citrusaurantium L. subsp. amara Engel that is mainly produced in France,Italy, Spain, Morocco and Algeria. Alternatively, orange flower concreteis obtained by extraction, and about 50% of an absolute are preparedwhen the concrete is extracted with alcohol. As the components containedtherein, l-linalol and linalyl acetate (the sum of these two ingredientsare about 35 to 40%), α-terpineol, geraniol, geranyl acetate, nerolidol(several %), and terpenes such as α-pinene, dipentene, camphene andocimene, and sulfur-containing compounds such as methyl anthranilate andindole are mentioned. Furthermore, for example, jasmone and benzaldehydeare contained in the concrete.

Bergamot oil is obtained by expression from pericarp of Citrus auratiumL. subsp. bergamia (Risso et Poit) Wright et Am. that is mainly producedin south Italy, Morocco, Tunisia, and Guinea. As the componentscontained therein, linalol and linalyl acetate are mentioned in a totalamounts of about 55 to 75%, and monoterpene hydrocarbon, nerol, citraland the like are contained. Furthermore, bergaptene, bergamotene andmethoxycoumarin are also contained as inherent components.

Mandarin oil is obtained by using sponge expression or expression frompericarp of Cirus reticulata Blanco var. “Mandarin”. When leaves of theplant is subjected to steam distillation, mandarin petitgrain oil isobtained. It comprises d-limonene as a main component,N-methylanthranilate methyl, citral, C₈ to C₁₁ of aliphatic aldehyde ofstraight chain and other terpenes. Leaf oil containsN-methylanthranilate methyl as the inherent component (50 to 60%).

Lemon oil is obtained by expression from pericarp of Citrus limone (L.),which is mainly produced in California, Sicily, Calabria, Spain andBrazil as well as is cultivated in Japan. It comprises, for example,d-limonene, citral, octyl aldehyde, nonyl aldehyde, linalol, andgeraniol.

Japanese pepper oil is obtained by homogenization and steam distillationfrom mature fruits of Xanthoxylum piperitum DC. As the componentscontained therein, dipentene (54%), geraniol and citronellol (10%),geranyl acetate and citronellyl acetate (20%), citronellal,β-phellandrene and Sanshol as the inherent components are mentioned.

Rutaceae oil is preferably used in combination of several kinds, andmore preferably three kinds. Lemon oil is preferably used as Rutaceaeoil.

The essentials oil containing the above-mentioned components areclassified into those mainly comprising phenolic compounds, those mainlycomprising sulfur-containing compounds, and those mainly comprisingterpene compounds. As an example of the oil comprising phenoliccompounds, thyme oil can be mentioned, and as examples of the oilcomprising sulfur-containing compounds, welsh onion oil and leek oil canbe mentioned. As examples of the oil comprising terpene compounds, therecan be mentioned Eucalyptus oil, lavender oil and Rutaceae oil.

Specifically, as examples of the phenolic compounds contained in suchoil, thymol or carvacrol in thyme oil can be mentioned. As examples ofthe sulfur-containing compounds, there can be mentioned diallylsulfide,diallyldisulfide, allicin and diallyltrisulfide, which are contained ingarlic oil. As examples of the oil comprising terpene compounds, therecan be mentioned l-camphene, d-camphene, β-eudesmol, dl-limonene (calledas dipentene), which are contained in Eucalyptus oil; and d-linalol,linalyl acetate, limonene, citral and terpineol, which are contained inlavender oil and Rutaceae oil. These compounds are fat-soluble compoundshaving a lower molecular weight in essential oil.

In the antioxidizing composition of the present invention, essential oilis used solely or in combination. Such essential oil may be combinedwith at least one purified compound as described above, and the compoundis not limited. As an example of the combination of essential oil withthe purified compound, there can be mentioned a combination of menthol,limonene and citral as purified compounds, with Eucalyptus oil, thymeoil and lemon oil; or with a mixed oil comprising Eucalyptus oil (40%),Rutaceae oil composed of three kinds of oil belong to Rutaceae oil(30%), Labiatae oil composed of two kinds of oil belong to Labiatae oil(25%) other than, and thyme oil (5%).

Since the essential oil described above is volatile, the composition ofthe present invention is also volatile.

The pharmaceutical composition of the present invention is prepared byadding suitable base material and additives to the antioxidizingcomposition. As the base material, a water absorptive acrylic resinhaving a high molecular weight, a porous substance, a polysaccharide andactivated charcoal are used.

As the water absorptive acrylic resin, which can absorb 400 to 800 timesin volume of water based on the volume of resin is preferably used.Specifically, there can be mentioned Aquafresh (purchased from SumitomoFine Chemicals Inc.) and Sunfresh (Sanyo Chemical Inc.). Such resin isadvantageously used because they function as a base material as well asthey have water-absorption ability to continuously regulate exothermicheat caused when exposed to water together with the polysaccharidedescribed below. Furthermore, they can adsorb and decompose basic aminegas, which is harmful for a living body, such as ammonia, trimethylamineand other amine gas.

The pharmaceutical composition may contain a coloring agent that absorbslight with a wavelength of 400 to 500 nm as an additive. As examples ofsuch coloring agents, there can be mentioned, indigo and coloring agentsabsorbing violet light. The above-mentioned coloring agents are used toabsorb the light having particular wavelength, and to utilize the energyof the light to enhance the energy level of molecules in the volatilecomposition. Then, vaporized molecules are transferred into the bodythrough absorption route as described below.

As the activated charcoal, which is finely divided particle,commercially available one, for example, Kayamax (Nippon Kayaku Co.) maybe used. The activated charcoal is adsorbed onto the antioxidizingcomposition, which is in the form of an oil layer on the surface of thethermoplastic resin, to form a capsule. The activated charcoal coversthe surface of the antioxidizing composition to prevent the interactionbetween such antioxidizing compositions and to control the exothermicheat.

As an example of the porous substance, zeolite, more specifically,Zeolum (purchased from Tosoh Corp.) may be mentioned. The poroussubstance preferably has pore size of 3 to 10 Å, and more preferably 3to 4 Å. Zeolite adsorbs moisture in the air to desorb the antioxidizingsubstances adsorbed on the base material by utilizing the adsorbingenergy thereby to maintain the residence time of the antioxidizingsubstances at the applied sites. The porous substance having theabove-mentioned pore size does not adsorb gasses except ammonia andwater vapor.

As examples of the polysaccharide, cellulose or chitosan are mentioned.Commercially available cellulose may be used, and chitosan may bepurchased from Koyo Chemicals, Inc. Polysaccharide has a function ofregulating exothermic heat together with the above-mentioned polymerresin, in the pharmaceutical compositions of the present invention.

Furthermore, as an example of the thermoplastic resin, polyethylene canbe mentioned, and as that of the water-soluble resin, polyvinyl alcoholcan be mentioned. Specifically, powdered polyethylene (Sumitomo FineChemicals Inc.) and Goserun (Nippon Synthetic Chemicals Inc.) can bementioned. These kinds of resin function as a carrier for adsorbing theantioxidizing compositions. Furthermore, they have a function of thatbinding the components of capsule, i.e. activated charcoal, basematerial and additives as a whole.

When the above-mentioned resin is used as the adsorbing carrier orbinder, the same resin can be used for both functions, or differentkinds of resin may be used.

The pharmaceutical composition of the present invention is preferablyprepared as formulation for parenteral administration, because itcomprises fat-soluble compounds having a low molecular weight and it isintroduced into lymphatic duct, and more preferably as percutaneousformulation.

As percutaneous routes for absorbing drugs, the following routes arementioned: a route which passes through corneum to reach capillaries inpapillary layer in subcticular bindweb, and a route which passes throughsebaceous gland of attached organs to reach blood vessels or lymphaticducts. In either route, drugs are delivered through capillary orcapillary lymphatic ducts into systemic circulation to express theirpharmacological action. When drugs are absorbed through percutaneousroute, lymphotropic activity of the drug is higher when they areadministrated via percutaneous route than per os or parenteral route,although their absorption rate varies depending on the structure of themor physicochemical properties.

Skin provides a route to excrete metabolites out of body, and itfunctions as barrier to prevent the invasion of extraneous material, andit is composed of epiderm and corium. Sebaceous membrane is formed oncorneum of epiderm, and corium has papipallry layer and reticular layer.

Sebaceous membrane on epiderm is the first absorption surface for theessential oil, and sebaceous membrane is an emulsion membrane. Sebaceousmembrane prevents invasion of bacteria, and regulates the bodytemperature. When the body temperature increases, the emulsion membranebecomes oil-in-water (o/w) type to accelerate water vapor to lower thetemperature by heat of vaporization. In contrast, when the prevention ofthe body temperature loss is intended, the membrane becomes water-in-oil(w/o) type to prevent sweating. Since the absorption of the essentialoil on the emulsion membrane is prevented when the membrane is (o/w), itmust be (w/o). Therefore, the alcohol type of components in theessential one mixed to accelerate vaporization of water on the surfaceof epiderm to enhance drug absorption on the membrane.

In papillary layer, artery-vein inosculation of peripheral vessels andcapillary lymphatic ducts are incorporated, and substance exchange orgas exchange is carried out. In such substance exchange field, thereexist carrier substances for drug such as lipid or protein, and theessential oil, which passes through sebaceous membrane to reach insideof papillary layer, flows into veins or lymphatic ducts. Then the drugis delivered to lymph nodes or peripheral tissues through veins orlymphatic ducts or both, and they are incorporated into cells via theirlipid bilayer in deliver or terminal of delivery.

The pharmaceutical composition of the present invention is formulated asfollows: the antioxidizing composition is adsorbed on a polymer resin toform an oil film, and then charcoal powder is absorbed on the oil filmto obtain a granule having an outer surface made of the charcoal. Thusobtained dust granule is inserted between two sheet materials, and alledges are bound by heat pressing to form a percutaneous agent.

The above-mentioned sheet material is preferably composed of paper ornon-woven fabric, and more preferably non-woven fabric, becausevaporized molecules in essential oil easily permeates the sheet materialwhen the components in the essential oil become gaseous state.

The pharmaceutical composition comprising the antioxidizing compositionthus obtained as the percutaneous sheet material is administrated byallowing it to contact with skin. For example, the percutaneous sheetmaterial is fastened by appropriate means, for example, double-sidedpressure sensitive adhesive tape, on the back center of a collar of ashirt for human to allow the sheet material to contact with back ofcervix. By fastening the sheet material as described above, thevaporized compositions of the present invention reaches the skin throughthe non-woven fabric, to be absorbed via sebaceous membrane. Then theyare transferred into blood vessels or lymphatic ducts. In order toadministrate to animals, for example, the sheet material is fastenedinside of a collar by using the double-sided pressure sensitive adhesivetape to be administrated percutaneously from the back cervix in the samemanner as for human. In human, the sheet material may be allowed totouch shoulder; hip or any other sites other than neck by fastening thesheet material with suitable means inside of clothes to administrate.

The fastened percutaneous sheet material may be exchanged every coupleof days to maintain a certain effect.

As described above, since the pharmaceutical composition of the presentinvention is lymphotropic, it is effective for aquired immunodeficiencysyndrome (AIDS) or autoimmune disease. It is known that HIV infects Tcell in charge of cellular immunity, that is helper T cell to decreaseimmunity. CD4, which is a firstly reported as human helper T cellmarker, is a glycoprotein expressed on T cell. T cells, which expressonly CD4 occupies about 65% of the peripheral T cells and they arereferred to as CD4+ T hereinbelow, have mainly helper T cell properties.Since CD4+ binds to HIV, it is known that CD4 functions as a receptor.

HIV infected T cell expressing CD4 has 100 times higher sensitivity thannormal cells, and therefore these cells generate active oxygen, that isfree radical.

Since free radical decreases the number of normal helper T cells toincrease relatively that of suppressor T cells, this leads to a decreasein the ratio of helper T cell number/suppressor T cell number toprogress AIDS. In such situation appeared, it generates both qualitativeand quantitative abnormalities in cytokine network, and promoteshistodialysis in lymph node, which is referred to as reactivehyperplasia.

When the pharmaceutical composition of the present invention isadministrated percutaneously to the patients or patient animals with theabove-mentioned symptoms, the essential oil contained in thepharmaceutical compositions make HIV infected CD4+ T cell cytostatic.Accordingly, such essential oil components are introduced into lymphaticducts to deliver lymph nodes. Then the components prevent furtherhistodialysis of lymphatic tissues, and they maintain the cells incytostatic situation so as to function the repair and regenerationability of the body itself. Therefore, resistance to the AIDS ofpatients or patient animals is enhanced. That is, the pharmaceuticalcomposition of the present invention has also immunopotentiationactivities.

Furthermore, since the administration of the pharmaceutical compositionof the present invention can be easily done by attaching the sheet formagent, and the administration can be easily stopped by detaching thesheet material from the fastened site. Only the essential oil derivedfrom natural sources or their components are vaporized to be transferredin the body, any serious side effects are not observed.

EXAMPLES

The present invention will now be described by the following examples indetail. However, the present invention is not limited to the examples.

Example 1

(1) Preparation of Antioxidizing Composition-1

An antioxidizing composition-1 of the present invention was prepared bymixing the essential oils in the amounts shown in Table 2 as theprincipal constituent. All flavors and fragrances used here werepurchased from Ogawa Perfumes Inc.

TABLE 2 Recipe of antioxidizing composition-1 Kinds of materials weight(%) Menthol 19.8 Limonene 3.2 Citral 1.6 Eucalyptus oil 5.6 Thyme oil1.6 Lemon oil 1.6 Total 33.4

(2) Preparation of Pharmaceutical Compositions Comprising theAntioxidizing Composition-1.

The pharmaceutical composition of the present invention is prepared bymixing the antioxidizing composition-1 with principle constituents inamount weight shown in Table 3.

TABLE 3 Pharmaceutical composition comprising the antioxidizingcomposition-1 Materials weight (mg) Principal constituent 33.4 Sunfresh(Sumitomo Fine Chemical Inc.) 55.6 Zeolum (Tosoh Corp.) 15.8 Chitosan(Koyo Chemical Co.) 8 Kayamax (Nippon Chemical Inc.) 39.6 Indigo tracePowdery polyethylene (Sumitomo Fine Chemical Inc.) 47.6 Total 200.0

Each essential oil shown in the above-mentioned (1) was respectivelymixed with the powdery polyethylene resin to form oil film on the resinsurface. Then the resin covered with essential oil and activatedcharcoal were mixed together to absorb the charcoal to form acarbon-coated capsule.

Separately from that, Sunfresh, Zeolum and Chitosan were mixed to form abase material. Then the carbon-coated capsule containing the essentialoil, which was made by the above-mentioned procedure, was added to thebase material together with indigo, and mixes to prepare thepharmaceutical composition of the present invention.

Two hundreds mg of thus obtained pharmaceutical composition of thepresent invention was covered with a piece of cotton cloth, and thensandwiched between two non-woven sheet materials. Then, all peripheraledges of the sheet materials were bonded by heat pressing to form apercuateous absorption agent.

Example 2

(1) Preparation of Antioxidizing Composition-2

An antioxidizing composition-1 of the present invention was prepared bymixing the essential oils in amount shown in Table 4 as the principalconstituent. All flavors and fragrances used here were purchased fromOgawa Perfumes Inc.

TABLE 4 Recipe of antioxidizing composition-2 Essential oil Ratio (%)weight (mg) Eucalyptus oil 40 0.44 Rutaceae oil 30 0.33 Labiatae oil 250.275 Thyme oil 5 0.055 Total 100 1.1

(2) Preparation of Pharmaceutical Compositions Comprising AntioxidizingComposition-2

The pharmaceutical composition of the present invention is prepared bymixing the antioxidizing composition-2 with principle constituents inamounts shown in Table 5.

TABLE 5 Pharmaceutical composition comprising antioxidizingcomposition-1 Materials weight (mg) Principal constituent 1.1 Watersoluble resin having high molecular weight 52.1 Polyvinyl alcohol 83.5Synthetic Zeolite 10.3 Activated charcoal 5.3 Thermoplastic resin 52.1L-Menthol 15.6 Total 220

Each essential oil shown in the above-mentioned (1) was respectivelymixed with the powdery polyethylene resin to form oil film on thesurface of the resin. Then the resin covered with essential oil and theactivated charcoal were mixed together adsorb the charcoal to form acarbon-coated capsule.

Separately from that, Aquafresh, Zeolum and PVA were mixed to form abase material. Then the carbon-coated capsule containing the essentialoil, which was made by the above-mentioned procedure, was added to thebase material, and mixed to prepare the pharmaceutical composition-2 ofthe present invention.

Two hundreds and twenty mg of thus obtained pharmaceutical compositionof the present invention was covered with a piece of cotton cloth, andthen sandwiched between two non-woven sheet materials. Then, allperipheral edges of the sheet materials were bonded by heat pressing toform the percutaneous absorption agent.

Example 3 Test Result for FIV Spontaneously Infected Cats

(1) Animals

As an experiment animal model for AIDS, a feline immunodeficiency virus,which is abbreviated as FIV, infected cat appearing AIDS (n=1), and twoFIV infected cats but not appearing AIDS (n=2) were used.

(2) Administration Procedure

The percutaneous absorption sheets prepared in the Preparation (2) werefastened inside of collars by using double-sided pressure sensitiveadhesive tape, and the collars were put on the neck of cats so as toallow the percutaneous sheets to touch the skin. The percutaneous sheetswere exchanged every three days.

(3) Administration Schedule

The sheet-form agents were administrated percutaneously for 4 weeks byfastening inside of the collars, and then nothing was administrated fornext 2 weeks. After that, a sheet-form placebo was administratedpercutaneously for 4 weeks, and then the agent was again administratedpercutaneously for 1 week.

Placebo was prepared from Sunfresh as the water-soluble resin havinghigh molecular weight, powdery polyethylene resin as thermoplasticresin, and Kayamax as activated charcoal. In the placebo, any essentialoil and a coloring agent are not contained.

(4) Items to be Measured

Items to be measured are the number of lymphocytes, the number of T cellexpressing sole CD4 (CD4+), the number of T cell expressing sole CD8(CD8+), the ratio of CD4+/CD8+, and IgG level.

(5) Result

Test results were shown in Table 5 and FIG. 1. As shown in Table 5 andFIG. 1, the number of lymphocytes, CD4+, CD8+ were increased, and theIgG level was enhanced during the agent was administrated. However, whenthe administration of the agent was stopped, those numbers and the levelwere decreased.

Example 4 Suppression of Disorder Caused by Active Oxygen

(1) Experiment Animals

Two groups of SPF cats are used as four per group.

(2) Administration Procedure

Percutaneous absorption agent prepared in the preparation (2) wasfastened inside of collars of the cats, and the collars were put on thenecks of them so as to allow the agent to touch the skin of back cervix.The agents were exchanged every three days.

(3) Administration Schedule and Evaluation

Treatment group received the fastened agent inside of the collar asdescribed above for two weeks. Control group received nothing.

Both groups received 1 mg/kg of methylene blue in every 8 hours, Heinzbody formation ratios in erythrocytes after 40 hours from methylene blueadministration were compared.

After methylene blue administration, placebo was administrated to thegroups in the same manner as the agent for four weeks. Then, the agentwas administrated for one week. As the placebo, a sheet without thecomposition of the present invention was used.

(4) Result

Results were shown in FIG. 2. As shown in FIG. 2, it was demonstratedthat Heinz body formation ratio was significantly suppressed, in thegroups received the composition of the present invention.

As described above, according to the present invention, there areprovided the antioxidizing composition comprising fat-soluble compoundshaving a low molecular weight in the essential oil, the pharmaceuticalcompound comprising antioxidizing composition and the process thereof.When the pharmaceutical composition is formulated to percutaneousabsorption agent, the composition is delivered to lymph throughsebaceous route to delete free radicals in lymph nodes, and increasesthe number of T cells expressing particular glycoproteins to showimmunopotentiation activity.

As mentioned above, it is demonstrated that the composition of thepresent invention is effective for immune diseases, especially AIDS.

INDUSTRIAL APPLICABILITY

The present invention is utilized in the field of pharmaceuticals.Particularly, essential oil derived from natural sources in thecomposition of the present invention is absorbed percutaneously to betransferred directly into a body, it is utilized for curing immunedisease, particularly AIDS.

What is claimed is:
 1. A sheet-form pharmaceutical preparation for gasdosage comprising a pharmaceutical composition-containing mixture whichis sandwiched between two sheet-form materials; said mixture comprising(i) a carbon-coated pharmaceutical composition comprising at least threekinds of carbon-coated particles (A), (B) and (C), wherein (A) isprepared by allowing a water-insoluble powdery thermoplastic resin toadsorb one essential oil selected from Rutaceae plant oil group (a)consisting of lime oil, orange oil, grapefruit oil, bergamot oil,mandarin oil, lemon oil, which are obtained by expression; and nerolioil and Japanese pepper oil, which are obtained by seam distillation,and then coating the essential oil-adsorbed powdery thermoplastic resinwith finely divided activated carbon particles; (B) is prepared byallowing a water-insoluble powdery thermoplastic resin to adsorb oneessential oil selected from Labiatae plant oil group (b) consisting ofPerilla oil, Agastache rugosa O, Kuntze oil, Clary sage oil, Sage oil,Thyme oil, Nepeta oil, Japanese Mint oil, Peppermint oil, Spearmint oil,Pennyroyal oil, Patchouli oil, Rosemary oil, Basil oil, Lavandin oil,and Lavender oil, all of which are obtained by steam distillation, andcoated with finely divided activated carbon particles; (C) is preparedby allowing a water-insoluble powdery thermoplastic resin to adsorbessential oil selected from Myrtaceae plant oil group (c) consisting ofClove oil, Pimenta oil, Bay oil, Cineole-type Eucalyptus oil, geranylacetate-type Eucalyptus oil, and Citronellal-type Eucalyptus oil, all ofwhich are obtained by steam distillation, and coated with finely dividedactivated carbon particles, (ii) a water-absorptive resin having ahigh-molecular-weight which is capable of absorbing 400 to 800 times itsvolume of water based on the volume of the water-absorptive resin, (iii)a porous substance, (iv) a polysaccharide, and (v) said water-insolublepowdery thermoplastic resin or a water-soluble thermoplastic resin. 2.The pharmaceutical preparation for gas dosage as claimed in claim 1,wherein said mixture further comprises a coloring agent, which absorbslight with a particular wavelength.
 3. A sheet-form pharmaceuticalpreparation for gas dosage comprising a pharmaceuticalcomposition-containing mixture which is sandwiched between twosheet-form materials; said mixture comprising (i) a carbon-coatedpharmaceutical composition comprising at least three kinds ofcarbon-coated particles (A), (B) and (C) according to claim 1, (ii) awater-absorptive acrylic resin as a water-absorptive resin having ahigh-molecular-weight which is capable of absorbing 400 to 800 times itsvolume of water based on the volume of the water-absorptive acrylicresin, (iii) zeolite as a porous substance, (iv) chitosan as apolysaccharide, (v) powdery polyethylene as the water-insolublethermoplastic resin, and (vi) indigo as a coloring agent which absorbslight with a particular wavelength.
 4. A sheet-form pharmaceuticalpreparation for gas dosage comprising a pharmaceuticalcomposition-containing mixture which is sandwiched between twosheet-form materials; said mixture comprising, (i) at least three kindsof carbon coated particles, which are selected from the group consistingof (A), (B) and (C) according to claim 1, together with carbon coatedparticles which are prepared by allowing powdery polyethylene as awater-insoluble powdery thermoplastic resin to adsorb L-menthol, andthen coating the L-menthol-adsorbed powdery polyethylene with finelydivided activated carbon particles, (ii) a water-absorptive acrylicresin as a water-absorptive resin having a high-molecular-weight whichis capable of absorbing 400 to 800 times in volume of water based on thevolume of the water-absorptive acrylic resin, (iii) zeolite as a poroussubstance, and (iv) polyvinyl alcohol as a water-soluble thermoplasticresin.